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1 
By: Bijaya Kumar Uprety 
Fluorimetry
LUMINESCENCE SPECTROSCOPY 
•The emissionof radiation from a species after that species has absorbed radiation. 
LUMINESCEN...
What is luminescence ? 
Luminescenceistheemissionofphotonsfromelectronicallyexcitedstate. 
Luminescenceisdividedintotwotyp...
LUMINESCENCE SPECTROSCOPY 
Absorption first - Followed by emission in all directions , usually at a lower frequency 4
Types of luminescence 
(classification according to the means by which energy is supplied to excite the luminescent molecu...
LUMINESCENCE SPECTROSCOPY 
•Collectively,fluorescenceandphosphorescenceareknownasphotoluminescence. 
•Athirdtypeofluminesc...
Introduction 
•Alargenumberofsubstanceabsorbultravioletandvisiblelightenergy.But,therearesomesubstanceswhichloseexcessener...
Concept of singlet and triplet state 
Singlet and triplet states 
•Ground state –two electrons per orbital; electrons have...
9
Fluorimetry 
•Fluorimetryisananalyticalmethodforthemeasurementoffluorescence,baseduponemissionofabsorbedradiationbythemole...
•Thebasisoffluorimetryisthemeasurementoffluorescence.Drugswhichareintrinsicallyfluorescent,aredeterminedfluorimetrically. ...
Principle of fluorimetry 
•Amoleculeabsorbsincidentelectromagneticradiationsandgetexcited. 
•Itisunstableinitsexcitedstate...
•Orbital changes can be explained as: 
1.Fluorescence: 
Singlet state Singlet excited state singlet ground state 
pp* (...
Fluorescence related to concentration 
ThefluorescenceradiantpowerFisproportionaltotheabsorbedradiantpower. 
F=(Po–P) 
wh...
•wherekisaconstantequaltoPoln.Thus,whentheconcentrationsareverydiluteandnotover2%oftheincidentradiationisabsorbed,therei...
Po 
Concentration of fluorescing species 
Theoretical behavior of fluorescence as a function of concentration.
Factors affecting fluorescence 
1.Natureofmolecules: 
•Themoleculeswhichhavethetendencytoabsorbelectromagneticradiations(i...
3.Highmolecularweightcompounds: 
Ifanelectronofanatomwithhighmolecularweightisexcited,itexhibitsdecreasedfluorescence. 
4....
6.AffectofpHofthesolution 
•Dependingupontheacidityoralkalinity,asubstancecanbeionizedorunionizedandhence,canbefluorogenic...
9.Affectofintensityofradiation: 
•Radiationofadequateintensitymustbeusedtoinducefluorescence. 
•Highintensityradiationcau...
•Compoundswithmorethantwocyclicstructureexhibitsfluorescence.E.g. vitaminK,Nucleosides,purines. 
•Rigidmoleculeslikemetalc...
Instrumentation 
•Thebasicdesignofinstrumentationformonitoringmolecularfluorescenceandmolecularphosphorescenceissimilartot...
Instrumentation for fluorescence spectroscopy 
Power 
supply 
Source 
Excitation monochromator 
Emission monochromator 
De...
Schematic diagram of a typical spectrofluorometer.
1) Light sources 
a. Gas discharge lamps : 
Xenon arc lamp 
High pressure mercury vapor lamp 
b. Incandescent lamps : Tung...
Cross-sectional view of an interference filter
Properchoiceofprimaryandsecondaryfilterstoavoidinterferencefromanothersubstance:a)excitationspectra(bothsubstancesfluoresc...
3) Sample compartment 
Cuvettes or cells with area of 1 cm2 .Usually made up of; 
Quarz or fused silica ----200 nm~ 800 nm...
29
Types of Fluorescent Molecules 
•Experimentallyitisfoundthatfluorescenceisfavouredinrigidmolecules,eg.,phenolphthaleinandf...
Types of Fluorescent Molecules 
•ItisthoughtthattheextrarigidityimpartedbythebridgingoxygengroupinFluoresceinreducestherat...
Application 
1.Analysis of medicinal compound: 
•A number of drugs can be estimated using fluorimetry. 
•Fluorescence is m...
(b)Organicandinorganiccompoundscanbemadefluorogenicbychemicalchangesuchasoxidation.E.g.Diphenylhydantoin(phenytoin)isoxidi...
2.Analysisofinorganiccompounds 
a.EstimationofUraniuminSalts. 
b.Somenon-fluorescentinorganicionscanbemadefluorescentbycom...
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Fluorimetry

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Fluorimetry

  1. 1. 1 By: Bijaya Kumar Uprety Fluorimetry
  2. 2. LUMINESCENCE SPECTROSCOPY •The emissionof radiation from a species after that species has absorbed radiation. LUMINESCENCEFLUORESCENCEPHOSPHORESCENCESPECTROSCOPYCHEMILUMINESCENCE 2
  3. 3. What is luminescence ? Luminescenceistheemissionofphotonsfromelectronicallyexcitedstate. Luminescenceisdividedintotwotypes,dependinguponthenatureofthegroundandtheexcitedstates. Inasingletexcitedstate,theelectroninthehigherenergyorbitalhastheoppositespinorientationasthesecondelectroninthelowerorbital.Thesetwoelectronsaresaidtobepaired.Returntothegroundstatefromanexcitedsingletstatedoesnotrequireanelectrontochangeitsspinorientation. Inatripletstatetheseelectronsareunpaired,thatis,theirspinshavethesameorientation.Achangeinspinorientationisneededforatripletstatetoreturntothesingletgroundstate. diamagnetic S1 paramagnetic T1 So
  4. 4. LUMINESCENCE SPECTROSCOPY Absorption first - Followed by emission in all directions , usually at a lower frequency 4
  5. 5. Types of luminescence (classification according to the means by which energy is supplied to excite the luminescent molecule) 1)Photoluminescence: Molecules are excited by interaction with photons of radiation. Fluorescence: Prompt fluorescence: S1S0 + h The release of electromagnetic energy is immediate or from the singlet state. Delayed fluorescence: S1T1S1S0 + h This results from two intersystem crossings, first from the singlet to the triplet, then from the triplet to the singlet. Phospholuminescence:T1S0+ h A delayed release of electromagnetic energy from the triplet state. 2) Chemiluminescence: The excitation energy is obtained from the chemical energy of reaction. 3) Bioluminescence: Chemiluminescence from a biological system: firefly, sea pansy, jellyfish, bacteria, protozoa, crustacea. 4)Triboluminescence: A release of energy when certain crystals, such as sugar, are broken. 5)Cathodoluminescence: A release of energy produced by exposure to cathode rays 6)Thermoluminescence: When a material existing in high vibrational energy levels emits energy at a temperature below red heat, after being exposed to small amounts of thermal energy.
  6. 6. LUMINESCENCE SPECTROSCOPY •Collectively,fluorescenceandphosphorescenceareknownasphotoluminescence. •Athirdtypeofluminescence-Chemiluminescence-isbaseduponemissionoflightfromanexcitedspeciesformedasaresultofachemicalreaction. •Infavorablecases,luminescencemethodsareamongstsomeofthemostsensitiveandselectiveofanalyticalmethodsavailable. •DetectionLimitsareasageneralruleatppmlevelsforabsorptionspectrophotometryandppblevelsforluminescencemethods. •Mostchemicalspeciesarenotnaturallyluminescent. •Derivatisationreactionsareoftenavailabletoformluminescentderivativesofnon-luminescentcompounds. •However,thisextrasteplessenstheattractivenessofluminescencemethods. •Fluorimetryisthemostcommonlyusedluminescencemethod. •Thetermsfluorimetryandfluorometryareusedinterchangeablyinthechemicalliterature. 6
  7. 7. Introduction •Alargenumberofsubstanceabsorbultravioletandvisiblelightenergy.But,therearesomesubstanceswhichloseexcessenergyasheatthroughcollisionswithneighboringatomsormolecules. •Theenergyemittedbythesesubstanceshasawavelengthlargerthanthatofabsorbed.Thisprocessofemittingradiationwithlargerwavelengththanthatofabsorbedisknownasluminiscence. •Luminiscenceismainlyoftwotypes: 1.Fluorescenceand2.Phosphorescence. 7
  8. 8. Concept of singlet and triplet state Singlet and triplet states •Ground state –two electrons per orbital; electrons have opposite spin and are paired •Singlet excited state Electron in higher energy orbital has the opposite spin orientation relative to electron in the lower orbital •Triplet excited state The excited valence electron may spontaneously reverse its spin (spin flip). This process is called intersystem crossing. Electrons in both orbitals now have same spin orientation
  9. 9. 9
  10. 10. Fluorimetry •Fluorimetryisananalyticalmethodforthemeasurementoffluorescence,baseduponemissionofabsorbedradiationbythemolecules.Whenamoleculeabsorbsincidentelectromagneticradiations,itisexcitedtohigherenergylevel,whereitisunstable. •Therefore,itreturnstothegroundstatebyemittingtheabsorbedradiations.Whentheemissionoccursdirectlyfromsingletexcitedstatetosingletgroundstatewithoutanytransitionsandwithoutanychangeinspinorientation,itisreferredasfluorescence. •Andwhentheemissionoccurswithinafractionofsecondtofewdaysofabsorptionofradiationsduetotransitionfromsingletexcitedstatetotripletstateandthentosingletgroundstate,itisreferredasphosphorescence.Itinvolvesthechangeinspinorientationduringthetransitionfromexcitedsinglettotripletandfromtriplettosingletgroundstate. •Themovementofelectronsfromexcitedsingletstatetotripletstate,iefromunpairedelectronswithoppositespintounpairedelectronswithsamespinistermedasintersystemcrossing. •Fluorescenceandphosphorescencearecombinedlycalledphotoluminescence. •Theenergyemittedbythesemechanismhasawavelengthlargerthanthatofabsorbed. 10
  11. 11. •Thebasisoffluorimetryisthemeasurementoffluorescence.Drugswhichareintrinsicallyfluorescent,aredeterminedfluorimetrically. e.g.Quininesulfatein0.1Nsulfuricacid;Ergometrinein1%tartaricacidetc. 11
  12. 12. Principle of fluorimetry •Amoleculeabsorbsincidentelectromagneticradiationsandgetexcited. •Itisunstableinitsexcitedstateandtendstoreturntothegroundstatebyemittingradiation. •Fluorescencecanbereferredastheradiationsemittedfromanexcitedmoleculeintransitionfromsingletexcitedstatetosingletgroundstate. •Influorimetry,radiationsemittedareoflongerwavelengththanabsorbedradiations.Thisisbecause,whentheradiationsfallonthemolecule,vibrationoccurs(in10-13seconds),andhasanaveragelifeof10-9seconds.Duringthevibrationperiod,lossofenergyoccursduetointermolecularcollisionsandsomeenergyislosttosolventmolecules(moleculesofthesolventusedforthedissolutionofthesampleinfluorimetry).Hencetheemittedradiationsareoflongerwavelengthandhavelessenergy.(sinceEαc/λ) 12
  13. 13. •Orbital changes can be explained as: 1.Fluorescence: Singlet state Singlet excited state singlet ground state pp* (No change in spin) 2. Phosphorescence: Singlet state Singlet excited state Triplet state (2 unpaired electrons) singlet ground state pp* (No change in spin) Change in spin •In either fluorescence or phosphorescence the frequency of the emitted radiation is less than the frequency of incident radiation. The relationship is ; 0> fluor> phos Where,  = frequency 13
  14. 14. Fluorescence related to concentration ThefluorescenceradiantpowerFisproportionaltotheabsorbedradiantpower. F=(Po–P) where=fluorescenceefficiency,Po=incidentpower,P=transmittedpower TherelationshipbetweentheabsorbedradiantpowerandconcentrationcanbeobtainedfromBeer’slaw. P/Po=10–A=10–bCor,P=Po10–bC or,F=Po(1–10–bC)------------(1) This is the fluorescence law. WhenexpandingtheexponentialtermsandassumingbCtobe0.05orless, onlythefirsttermintheseriesissignificantandequationcanbewrittenas F=Po(lnbC)=kbC-----------(2)
  15. 15. •wherekisaconstantequaltoPoln.Thus,whentheconcentrationsareverydiluteandnotover2%oftheincidentradiationisabsorbed,thereislinearrelationshipbetweenfluorescentpowerandconcentration. •WhenbCisgreaterthanabout1.5,10–bCismuchlessthan1andfluorescencedependsdirectlyontheincidentradiationpower. F=Po •Fromequation2,wecouldinferthatfluorescenceintensityisdirectlyproportionaltoconcentration.Anotherimportantresultcanbeimpliedfromequation2wherefluorescenceisshowntobedirectlyproportionaltotheintensityoftheincidentbeam.Thissuggeststhataveryintenselightsourceisnecessaryforfluorescenceinstrumentation.Alsosubstancesoflargearepotentialfluorescentmoleculesandshouldbesoughforbetterresults. 15
  16. 16. Po Concentration of fluorescing species Theoretical behavior of fluorescence as a function of concentration.
  17. 17. Factors affecting fluorescence 1.Natureofmolecules: •Themoleculeswhichhavethetendencytoabsorbelectromagneticradiations(ieUV-Vis)canonlyexhibitfluorescence. •Unsaturatedmoleculeswithpibondsandgoodresonancestabilitycanexhibitfluorescence.E.g.alkeneswithconjugateddoublebond. •Saturatedmoleculeswithsigmabondsdonotexhibitfluorescence.E.g. aliphaticandsaturatedcyclicorganiccompounds. •Ingeneral,thegreatertheabsorbancyofamolecule,themoreintenseitsluminiscence. 2.Affectoffunctionalgroups:Functionalgroupsexhibitmarkedeffectonfluorescenceoftenly. •Electrondonatinggroupsimprovefluorescence.E.g.amine,hydroxy,andmethoxygroups. •Electronacceptinggroupsdeteriorateorcompletelydestroyfluorescence.E.g.carboxylic,nitroandazogroup,halidesetc 17
  18. 18. 3.Highmolecularweightcompounds: Ifanelectronofanatomwithhighmolecularweightisexcited,itexhibitsdecreasedfluorescence. 4.Effectofconcentration: •Thereexistsalinearrelationshipbetweentheconcentrationofsampleandfluorescenceuptoanabsorbancevalueof0.02.Thelinearityholdsgoodtoabout5%ofcaseswithabsorbancesupto0.05. •Indilutesolution,theradiationdistributeuniformlythroughoutthesolutionandgetabsorbeduniformlygivinghighintensefluorescence. •Inhighlyconcentratedsolution,upperlayersofthesolutionabsorbmoreradiationsandlessamountofradiatonsaretranserredtolowerlayers.Thus, thereisnouniformradiationsabsorptionwhichresultsindecreasefluorescence. •Inhighlyconcentratedsolution,intramolecularcollisionscauselossofvibrationalenergyandcertainamountoffluorescecewhichisemittedisreabsorbedresultingindecreaseinintensityoffluorescence.Thisiscalledasconcentrationquenching. 5.Effectofoxygen:Thereisdecreaseinfluorescenceinthepresenceofoxygendueto •Directconversionoffluorogenicmaterialintonon-fluorogenicmaterial. •Indirectlyduetoquenching.‘Quenchingreferstothedecreaseinintensityoffluorescenceasaresultofdecreaseinthesensitivityofconstituents. 18
  19. 19. 6.AffectofpHofthesolution •Dependingupontheacidityoralkalinity,asubstancecanbeionizedorunionizedandhence,canbefluorogenicornon-fluorogenic. e.g.Phenolinneutralandalkalinemediumundergoesionisationandgivesweakfluorescence,whereasinacidmediumitisunionisedandgivesintensefluorescence. 7.AffectofTemperatureandViscosity •Alterationintemperaturemayaffecttheconcentrationandviscosityofthesample. •Increaseintemperaturemayresultinincreaseinconcentrationanddecreaseinviscosityresultinginintermolecularcollisionsanddeactivation ofexcitedmoleculedestroyingfluorescence. •Somesubstancesmayexhibitfluorescenceattemperaturelowerthanroomtemperatureorinaviscoussolventorglassymatrix. 8.Affectofimpurities:Substancesotherthanthesolutemoleculesareimpuritiesandexhibitfluorescencequenching.E.g.iodideionisextremelyeffectivequencher. 19
  20. 20. 9.Affectofintensityofradiation: •Radiationofadequateintensitymustbeusedtoinducefluorescence. •Highintensityradiationcausesdecreaseinfluorescenceduetophotochemicalchange. •Lightofsinglewavelength,i.e.monochromaticlightshouldbeused,asenergyofradiationvarieswithwavelength. 10.ChemicalQuenching: Thiscanoccurintwoways; •Theexcitedmoleculetransfersitsfluorescentintensitytosurroundingmolecules,ionsorimpuritybyintercollisions,therebydestroyingfluorescence. •Theunexcitedmoleculemayformastablecomplexwithquenchermoleculeinhibitingexcitationandfluorescence. 11.Affectofstructureofthecompounds: •Closedringaromaticcompoundsexhibitfluorescencee.g.fluorescein, Eosin. 20
  21. 21. •Compoundswithmorethantwocyclicstructureexhibitsfluorescence.E.g. vitaminK,Nucleosides,purines. •Rigidmoleculeslikemetalcomplexeshaveenhancedfluorescenceastheyinhibittheliberationofexcitationenergy. •Thepositionoffunctionalgroup(chromophore)whichisresponsibleforabsorptionaffectsfluorescence. 21
  22. 22. Instrumentation •Thebasicdesignofinstrumentationformonitoringmolecularfluorescenceandmolecularphosphorescenceissimilartothatfoundforotherspectroscopies.Themostsignificantdifferencesarediscussedinthefollowingsections. •MolecularFluorescenceAtypicalinstrumentalblockdiagramformolecularfluorescenceisshowninFigure10.45.Incontrasttoinstrumentsforabsorptionspectroscopy,theopticalpathsforthesourceanddetectorareusuallypositionedatanangleof90°. •Twobasicinstrumentaldesignsareusedformeasuringmolecularfluorescence.Inafluorometertheexcitationandemissionwavelengthsareselectedwithabsorptionorinterferencefilters.Theexcitationsourceforafluorometerisusuallyalowpressuremercuryvaporlampthatprovidesintenseemissionlinesdistributedthroughouttheultravioletandvisibleregion(254,312,365,405,436,546,577,691,and773nm). •Whenamonochromatorisusedtoselecttheexcitationandemissionwavelengths, theinstrumentiscalledaspectrofluorometer.Withamonochromator,theexcitationsourceisusuallyahigh-pressureXearclamp,whichhasacontinuumemissionspectrum. 22
  23. 23. Instrumentation for fluorescence spectroscopy Power supply Source Excitation monochromator Emission monochromator Detector Sample cell Slit Data processor General layout of fluorescence spectrophotometer
  24. 24. Schematic diagram of a typical spectrofluorometer.
  25. 25. 1) Light sources a. Gas discharge lamps : Xenon arc lamp High pressure mercury vapor lamp b. Incandescent lamps : Tungsten wire filament lamp c. Laser : tunable dye laser d. X-ray source for X-ray fluorescence 2) Wavelength selection devices a. Filters : Absorption filters ---tinted glass or gelatin containing dyes sandwiched between glass Interference filters ---thin transparent layer of CF2or MgF2sandwiched two parallel, partially refelecting metal films b. Monochromators : Gratings Prism
  26. 26. Cross-sectional view of an interference filter
  27. 27. Properchoiceofprimaryandsecondaryfilterstoavoidinterferencefromanothersubstance:a)excitationspectra(bothsubstancesfluoresceoversamewavelengthregion,b) fluorescencespectra(bothsubstancesabsorbinsamewavelengthregion).
  28. 28. 3) Sample compartment Cuvettes or cells with area of 1 cm2 .Usually made up of; Quarz or fused silica ----200 nm~ 800 nm Glass or plastic ----300 nm~ 4) Detectors Photomultiplier tube
  29. 29. 29
  30. 30. Types of Fluorescent Molecules •Experimentallyitisfoundthatfluorescenceisfavouredinrigidmolecules,eg.,phenolphthaleinandfluoresceinarestructurallysimilarasshownbelow.However,fluoresceinshowsafargreaterfluorescencequantumefficiencybecauseofitsrigidity. • phenolphthalein
  31. 31. Types of Fluorescent Molecules •ItisthoughtthattheextrarigidityimpartedbythebridgingoxygengroupinFluoresceinreducestherateofnonradiativerelaxationsothatemissionbyfluorescencehassufficienttimetooccur. Fluorescein
  32. 32. Application 1.Analysis of medicinal compound: •A number of drugs can be estimated using fluorimetry. •Fluorescence is made to produce from non- fluorogenicdrugs by following ways: (a) Some drugs are capable of exhibiting fluorescence in an appropriate solvent. e.g. Quinine in 0.1 N Sulfuric acid, Riboflavin in 1% tartaric acid, Aminocrinein 0.1 N HCl. 32
  33. 33. (b)Organicandinorganiccompoundscanbemadefluorogenicbychemicalchangesuchasoxidation.E.g.Diphenylhydantoin(phenytoin)isoxidisedbyalkalineKMnO4toformbenzophenonewhichexhibitsflurescence. (c)Organicandinorganiccompoundsarecomplexedwithsuitablereagentstomakethemfluorogenic. (d)Whentwoormoredrugsarepresent,eachdrugcanbeestimatedindividuallybyadoptingsuitablemethodlike; Conversionofacidictoalkalinesolutionorviceversa. Conversionofionictonon-ioniccompoundorviceversa. Selectionofwavelengthofexcitationforeachdrug. Extractionofanyonedrugfromthemixtureandanalysingit. (e)Preparationoffluorogenicderivativefromnon-fluorogenicdrug.Someoftheexamplesinclude; Complexofatropinewitheosinissolubleinchloroformandexhibitfluorescence. Othernon-fluorogenicdrugswhichcanbeanalysedaremorphineandcodeine. 33
  34. 34. 2.Analysisofinorganiccompounds a.EstimationofUraniuminSalts. b.Somenon-fluorescentinorganicionscanbemadefluorescentbycomplexingitwithnon-fluorescentorganicreagents.Hencetheseelementcanbefluorimetricallyanalysed. 3.FluorescentindicatorsareusefulinfluorimetricdeterminationasthecolorandintensityoffluorescentdependsuponthepHofthesolution. 4.Hydrogenbonding,geometricalisomerism,polymerization,tautomerismandreactionratescanbestudiedfluorimetrically. 5.Fluorimetrycanbeusedforbothqualitativeandquantitativeestimationofsteroids,proteins,plantpigmentsetc 34

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